Cytoplasmic dynein, a multi-subunit complex, is a minus-end-directed microtubule motor. With its accessory complex, dynactin, cytoplasmic dynein performs multiple cellular functions including retrograde vesicle transport and organelle distribution. The molecular mechanisms involved in regulating the activity of cytoplasmic dynein are not well understood. Our long-term goal is to understand how the intracellular targeting and motor activity of cytoplasmic dynein is regulated in vivo by using the filamentous fungus Aspergillus nidulans as a genetic model system. We have identified multiple genes that function in the cytoplasmic dynein pathway through the genetic analyses of A. nidulans mutants defective in nuclear distribution (nud). While some nud genes encode components of the cytoplasmic dynein and dynactin complexes, novel regulators have also been discovered. For example, the nudF gene is homologous to Lis1, a human lissencephaly (smooth brain) disease gene involved in neuronal migration. We have recently found that GFP labeled cytoplasmic dynein, dynactin, and NUDF, all accumulate at the plus ends of microtubules in vivo. KINA, an A. nidulans homolog of the conventional kinesin, is required for the microtubule plus-end localization of dynein and dynactin, but not NUDF. Interestingly, the plus-end accumulation of dynein and dynactin increases in the absence of NUDF. Based on these and other results, we hypothesize that cytoplasmic dynein is transported by KINA to the microtubule plus end where it receives its cargo and is activated by NUDF/LIS 1 to depart from the plus end for the minus end. This application proposes experiments to test such a hypothesis and to characterize more regulators of cytoplasmic dynein function. Our first specific aim is to study the mechanism of KINA-dependent microtubule plus-end localization of cytoplasmic dynein, by using living cell imaging and photobleaching techniques, as well as by developing an in vitro system to test which proteins are sufficient for dynein or dynactin's microtubule plus-end localization.
Our second aim i s to examine dynein motor activity in mutants that lack NUDF and mutants that are defective in plus-end dynein localization, to determine whether cytoplasmic dynein motor activity is dependent upon its plus-end localization and the presence of NUDF.
Our third aim i s to create null-mutants of several proteins in the dynein complex to study their functions in dynein regulation, and identify novel dynein regulators by cloning additional nud genes. ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM069527-03
Application #
7002232
Study Section
Cell Development and Function Integrated Review Group (CDF)
Program Officer
Rodewald, Richard D
Project Start
2004-01-02
Project End
2007-12-31
Budget Start
2006-01-01
Budget End
2006-12-31
Support Year
3
Fiscal Year
2006
Total Cost
$255,819
Indirect Cost
Name
Henry M. Jackson Fdn for the Adv Mil/Med
Department
Type
DUNS #
144676566
City
Bethesda
State
MD
Country
United States
Zip Code
20817
Zhang, Jun; Yao, Xuanli; Fischer, Lauren et al. (2011) The p25 subunit of the dynactin complex is required for dynein-early endosome interaction. J Cell Biol 193:1245-55
Zhang, Jun; Tan, Kaeling; Wu, Xufeng et al. (2011) Aspergillus myosin-V supports polarized growth in the absence of microtubule-based transport. PLoS One 6:e28575
Zhang, Jun; Zhuang, Lei; Lee, Young et al. (2010) The microtubule plus-end localization of Aspergillus dynein is important for dynein-early-endosome interaction but not for dynein ATPase activation. J Cell Sci 123:3596-604
Zhai, Bing; Zhou, Henry; Yang, Liangpeng et al. (2010) Polymyxin B, in combination with fluconazole, exerts a potent fungicidal effect. J Antimicrob Chemother 65:931-8
Zhang, Jun; Li, Shihe; Musa, Shamsideen et al. (2009) Dynein light intermediate chain in Aspergillus nidulans is essential for the interaction between heavy and intermediate chains. J Biol Chem 284:34760-8
Zhang, Jun; Wang, Liqin; Zhuang, Lei et al. (2008) Arp11 affects dynein-dynactin interaction and is essential for dynein function in Aspergillus nidulans. Traffic 9:1073-87
Zhuang, Lei; Zhang, Jun; Xiang, Xin (2007) Point mutations in the stem region and the fourth AAA domain of cytoplasmic dynein heavy chain partially suppress the phenotype of NUDF/LIS1 loss in Aspergillus nidulans. Genetics 175:1185-96
Efimov, Vladimir P; Zhang, Jun; Xiang, Xin (2006) CLIP-170 homologue and NUDE play overlapping roles in NUDF localization in Aspergillus nidulans. Mol Biol Cell 17:2021-34
Li, Shihe; Oakley, C Elizabeth; Chen, Guifang et al. (2005) Cytoplasmic dynein's mitotic spindle pole localization requires a functional anaphase-promoting complex, gamma-tubulin, and NUDF/LIS1 in Aspergillus nidulans. Mol Biol Cell 16:3591-605